Heme-linked protonation of HCN, CO, NO and O2 complexes of reduced horseradish peroxidases.

Small reversible changes in the absorption spectra of HCN, CO, NO and O₂ complexes of ferrous diacetyldeuteroperoxidase A, not hitherto observed, were attributed to proton dissociation of a distal amino acid residue. From spectrophotometric titration data the pK_a was measured as 5.5 (HCN), 5.6 (ligand free), 6.0 (CO), 6.55 (NO) and 8.0 (O₂). The value of 8.0 for the pK_a of the O₂ complex was also obtained from a curve of pH dependence of proton uptake in the reaction of the ferrous enzyme with O₂. Absorption bands in the visible region were shifted to longer wavelengths in the order of CO to NO to O₂ which is the decreasing order of the energy of π¹ level of these diatomic ligands.The pK_a values for CO complexes of ferroperoxidases, isoenzymes A and (B+C) were varied with substituents at the 2 and 4 positions of deuterohemin IX, and the $\text{Δp}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{Δp}\text{K}{}_3$ ratio was about 0.3 in both series of isoenzyme preparations, where pK₃ is a measure of basicity of pyrrole nitrogen.The present data support the previous conclusion (Yamada and Yamazaki (1974) Arch. Biochem. Biophys., 165, 728) that the pK_a for ferroperoxidases, measured from small reversible changes in the absorption spectra, represents a proton dissociation constant of a distal amino acid residue and that there is hydrogen bonding between the residue and a ligand atom directly bound to the iron atom.